Infrared imaging of small molecules in living cells: from in vitro metabolic analysis to cytopathology

Abstract

A major topic in InfraRed (IR) spectroscopic studies of living cells is the complexity of the vibrational spectra, involving hundreds of overlapping absorption bands from all the cellular components present at detectable concentrations. We focus on the relative contribution of both small-molecule metabolites and macromolecules, while defining the spectroscopic properties of cells and tissue in the middle IR (midIR) region. As a consequence, we show the limitations of current interpretative schemes that rely on a small number of macromolecules for IR band assignment. The discussion is framed specifically around the glycolytic metabolism of cancer cells because of the potential pharmacological applications. Several metabolites involved in glycolysis by A549 lung cancer cells can be identified by this approach, which we refer to as Correlated Cellular Spectro-Microscopy (CSM). It is noteworthy that the rate of formation or consumption of specific molecules could be quantitatively assessed by this approach. We now extend this analysis to the two-dimensional case by performing IR imaging on single cells and cell clusters, detecting variations of metabolite concentration in time and space across the sample. The molecular detail obtained from this analysis allows its use in evaluating the pharmacological effect of inhibitors of glycolytic enzymes with potential consequences for in vitro drug testing. Finally we highlight the implications of the spectral contribution from cellular metabolites on applications in IR spectral cytopathology (SCP).